The immune system has evolved to process and recognize intracellular antigens via a class I MHC mediated antigen presentation pathway. Class I MHC restricted antigens are targeted by CD8 positive T cells largely consisting of cytotoxic T cells (CTL). An essential property of tumor antigen recognition by CD8 positive CTL cells is the requirement of the TCR to engage class I MHC/peptide complexes. Class I MHC restricted peptides are customarily derived from intracellular proteins. Thus, stimulation of immunity following in vivo administration of recombinant proteins preferentially stimulates antibody responses and only weak CTL responses.
Based on these fundamentals of CTL recognition, several methods have traditionally been utilized to stimulate CTL responses. Peptides with conanical sequences optimized for MHC class I binding can displace non-covalently bound peptide on cell surface MHC class I in a concentration dependent manner. Peptides have successfully been utilized to stimulate CTL responses in vivo but generally require the use of adjuvants such as IFA. Alternatively, genetic approaches such as viral vectors or naked DNA have been utilized to introduce gene sequences directly into cells to expression intracellularly to deliver antigen directly into the endogenous class MHC antigen processing machinery. However, a limitation to targeting specific antigens limited to a relatively small subset of tumors is that mutations or overexpression of specific tumor associated antigens must be determined and applied on an individual basis.
An optimal immunotherapeutic strategy would allow treatment of a broad spectrum of human malignancies with a common pharmaceutical product. Stimulating immune responses to tumors with p53 mutations may enable treatment of a broad spectrum of tumors as approximately 50% of tumors have mutations in the p53 tumor suppresser gene. The p53 tumor associated antigen is characterized as a mutant TAA. Initially, strategies were designed to elicit CTL responses to xe2x80x9cunique peptide antigensxe2x80x9d generated by the p53 mutant sequences. This strategy was based on the premise that tumor specific CTL recognize peptides, derived from endogenously synthesized cellular proteins, presented by class I major histocompatibility complex (MHC) molecules. However, targeting such tissue-specific antigens may restrict immunotherapies to a very limited set of tumors as the mutations occur in many different loci within the p53 gene.
A distinguishing characteristic of p53 tumor associated antigens is that mutations within the p53 tumor associated antigen occur in approximately 50% of human malignancies. Moreover, most of the mutations result in an increase in the half-life of the p53 proteins resulting in a marked overexpression in tumor cells. The extended expression of p53 in tumor cells may modify processing and presentation of wt p53-derived peptides by MHC class I molecules. Since most MHC class I receptors are occupied by endogenously derived cellular proteins, a shift in p53 expression may result in a disproportionate number of MHC molecules presenting p53 derived peptides. This may permit development of T cell responses to p53 by T cells expressing T cell receptors (TCR) with low to moderate affinity. Indeed, several lines of evidence support the premise that tolerance to p53 can be overcome resulting in an immune response to tumor cells overexpressing p53. Anti-p53 antibodies have been found in sera of patients with several types of cancers and T cell lymphoproliferative responses to p53 have been detected in breast cell cancer patients. Moreover, subcutaneous immunization with canarypox viral vectors expressing p53 protected mice from challenge with tumors overexpressing p53.
Another distinguishing feature of utilizing p53 transgenes to stimulate tumor immunity is that transduction of tumors cells bearing p53 mutations with wt p53 transgenes generally induces apoptosis. Recent reports indicate that phagocytosis of apoptotic cells by dendritic cells may be an important mechanism for antigen transfer to dendritic cells and subsequent stimulation of specific T cell immunity to antigens expressed by the apoptotic cell. Thus, the induction of apoptosis may promote antigen loading of unknown TAA following phagocytosis by dendritic cells resulting in a more broad spectrum of tumor associated antigenic stimuli as described in the methods of use section below.
An important issue for strategies to induce immunity to self antigens that are differentially expressed in tumor cells is to assess the potential for autoimmune responses to normal cells. Several preclinical studies in mice induced to respond to p53 using peptide antigens and viral vectors have been reported in which specific cytotoxic T cell responses to tumor cells overexpressing p53 were observed while cells expressing normal levels of p53 were not killed. This has been attributed to the induction of T cell responses by CTL cells bearing T cell receptors (TCR) with low to moderate affinity for self MHC/p53 peptide complexes; T cells bearing TCR with high affinity are presumed to be eliminated during thymic negative selection. Moreover, in recent clinical trials, autoimmune responses were assessed in patients undergoing ex vivo dendritic cell immunotherapy to induce immune responses to melanoma antigens. Specific responses to melanoma antigens were successfully induced in the patients, yet no clinically overt sign of autoimmunity were observed.
Genetic immunization has proven to be an effective means to stimulate CTL mediated immunity to viruses and tumors. Expression of transgenes or minigenes (encoding antigenic peptides) intracellularly results in delivery of the immunogen directly into the MHC class I peptide processing and antigen loading system. Moreover, the entire antigenic protein or multiple proteins can be expressed allowing natural processing and loading of the antigenic peptides onto numerous allelic MHC class I and class II molecules permitting a more broad immune response. DNA vaccines and viral vectors show significant promise as effective vehicles for genetic immunization.
In vivo CTL activation may be mediated by dendritic cells. Dendritic cells are professional antigen presenting cells. Dendritic cells exist in distinct functional states. Immature dendritic cells corresponding to those found in peripheral tissues, exhibit a phenotype in which most class II molecules are intracellular and localized to lysosomes. In this phase, they are capable of uptake of antigen. These immature dendritic cells xe2x80x9cpatrolxe2x80x9d the peripheral tissues in search of foreign antigens. Culturing dendritic cell precursors in vitro with GM-CSF and IL-4 induces differentiation into immature dendritic cells capable of highly efficient antigen uptake. Further differentiation into mature DC with highly developed antigen presentation functions can be induced by CD40 ligation, TNFxcex1 or LPS. Moreover, antigen pulsed dendritic cells under these culture conditions are extremely efficient at stimulating lymphoproliferative and CTL responses both in vitro and in vivo (Current estimates indicate that dendritic cells may be 10-100 times more potent as antigen presenting cells than other APCs such as macrophage and B cells). These immature dendritic cells mature into an intermediate phenotype in which intracellular class II molecules are found in peripheral non-lysosomal vesicles. The intermediate cells then differentiate into late dendritic cells which express almost all of their class II molecules on the plasma membrane. The maturing dendritic cells migrate to the lymph nodes where they present the processed peptides to T-cells.
The ability of dendritic cells to present tumor antigens to the immune system to recruit an immune response to tumor cells has been suggested as an anticancer therapy. The majority of dendritic cell therapeutic studies to date have utilized ex vivo strategies to load dendritic cells with antigen and to activate them to stimulate T cell immunity following reinfusion. The antigen pulsed dendritic cells are then reinfused into the hosts by various different routes to stimulate tumor specific T cell immunity. One advantage of ex vivo dendritic cell therapeutic strategies is the broader capacity to charge dendritic cells with various forms of antigen in vitro in manners not feasibly done in vivo (i.e., pulsing with recombinant protein antigens, tumor cell lysates or even tumor RNA extracts). This suggests that dendritic cells may be special in their capacity to process antigens using alternative antigen processing systems. Genetic methods using various viral vectors have also been successfully utilized to transduce dendritic cells with transgenes encoding tumor associated antigens or minigenes encoding MHC restricted peptide sequences.
A preferred strategy would be to induce direct in vivo stimulation of dendritic cells to induce strong tumor immunity and the preferred procedures in this invention utilize strategies to stimulate dendritic cell mediated immunotherapy in vivo. Genetic antigen delivery systems are well suited for in vivo strategies. However the use of genetic adjuvants such as cytokines, to mobilize dendritic cells and induce differentiation and activation, as well as chemokines to direct immature dendritic cells to the tumor site and mature dendritic cells to the lymph nodes may be essential to achieve consistently effective anti-tumor immunity and regression. The invention described herein are designed to effectively induce strong inflammatory responses mediated by dendritic cells within the tumor to enable the immune response to overcome the natural tolerance mechanisms utilized be tumors to evade immune surveillance and or clearance of tumors.
The present invention provides compositions which are engineered to induce killing of tumor cells and concomitantly mobilize differentiate, activate and attract dendritic cells through the expression of cytokines and dendritic cell chemoattractants. This combination therapy is called p53 adjunctive immunotherapy. This invention is designed to effectively induce multiple stages of dendritic cell differentiation, activation and migration in vivo using gene therapy delivery systems. First, recruitment of immature dendritic cells (that efficiently phagocytose and process tumor antigens) to the site of the tumor. Second, promote activation and migration of the dendritic cells to regional lymph nodes where the activated dendritic cells can stimulate tumor specific T cells and overcome tolerance to stimulated strong tumor immunity. The transduction of tumor cells with p53 is designed to reduce the primary tumor mass via cell cycle arrest and apoptosis as well as to promote antigen transfer of unknown or cryptic tumor antigens to dendritic cells by means of phagocytosis of the apoptotic tumor cells. Thus, providing a means to generate diverse immune responses to a broad spectrum of multiple tumor associated antigen, which may be essential for effective tumor immunity to heterogeneous tumor cells in the primary tumor and distal metastases. Moreover, this invention describes the rational design of utilizing viral vectors (preferred vector is rAd) for multiple administrations of targeted delivery to dendritic cells which can promote differentiation and activation of the transduced dendritic cells (thus augmenting in vivo stimulation of T cells, NK cells and B cells. The present invention provides a method to induce an anti-tumor immune response through the use of such compositions.